Sialic acid/fucose based medicaments

ABSTRACT

Compounds that are synthetically inexpensive to make relative to the naturally occurring selectin ligands and that retain selectin binding activity are described that have a three-dimensionally stable configuration for sialic acid and fucose, or analogs or derivatives of these groups, such that sialic acid and fucose are separated by a non-carbohydrate linker that permits binding between those groups and the selectins, such compounds being represented by the following general structural formula I(a): ##STR1## wherein m and n are independently an integer of from 1 to 5, Y and Z are independently a connecting moiety selected from the group consisting of --CH 2  --, --O--, --S--, --NR&#39; and --NR&#39;R&#34;-- (wherein R&#39; and R&#34; are independently H or an alkyl containing 1 to 4 carbon atoms); X is a connecting moiety which is selected from the group consisting of --O--, --S-- and --N--; and --R&#39;&#34; may be --R&#34; or any moiety which does not interfere with the three-dimensional configuration of A or B so as to interfere with selectin binding and is preferably a moiety selected from the group consisting of --OR&#34;, --SR&#34;, --I, --N 3 , and --NR&#39;R&#34;, and A is selected from the group consisting of α and β forms of sialic acid, Kemp&#39;s acid, Quinic acid, Glyceric acid, Lactic acid and acetic acid, and esters thereof and B is selected from the group consisting of α and β forms of L-Fucose and esters and substituted forms thereof wherein one or more of the --OH groups is independently --F, or --NR IV , R V  wherein R IV  and R V  are independently an alkyl contain 1 to 5 carbons.

This application is divisional of application Ser. No. 08/078949 filedJun. 16, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of medicinal chemistry,and specifically to medicaments that are characterized by their capacityto bind to one or more of the three known selectins; LECAM-1, LECAM-2,or LECAM-3. The medicaments consist of three chemical moietiescovalently linked in the following order: sialic acid, or an analogue orderivative thereof, a non-saccharide spacer, and fucose or an analogueor derivative thereof. Such medicaments have significant applicationsfor diagnosis or prophylactic or therapeutic treatment of certaindiseases including cancer, autoimmunity, and inflammation.

BACKGROUND OF THE INVENTION

A large body of data has been accumulated that establishes a family ofreceptors, the selectins (hereinafter LECAMs) in certain diseasesincluding cancer, autoimmunity, and in the inflammatory response. Thethree known members of this family, L-Selectin (LECAM-1, LAM-1,gp90MEL), E-Selectin (LECAM-2, ELAM-1) and P-Selectin (LECAM-3, GMP-140,PADGEM), each contain a domain with homology to the calcium-dependentlectins (C-lectins), an EGF-like domain, and several complement bindingprotein-like domains (Bevilacqua et al., Science (1989) 243:1160-1165;Johnston et al., Cell (1989) 56:1033-1044; Lasky et al., Cell (1989)56:1045-1055; Tedder et al., J. Exp. Med. (1989) 170:123-133). It hasbeen proposed that the selectins bind to particular ligands and thatthis accounts for their biological activity. Thus, drugs that interferewith or prevent binding of the ligands to the selectins will be usefulmedicaments for treating a variety of diseases.

For instance, adhesion of circulating neutrophils to stimulated vascularendothelium is a primary event of the inflammatory response. P-selectinhas been shown to be centrally involved particularly as related to acutelung injury. Mulligan et al. have reported strong protective effectsusing anti-P-selectin antibody in a rodent lung injury model. (Mulligan,M. S. et al., J. Clin. Invest. (1991) 90:1600).

ELAM-1 is particularly interesting of the three selectins because of itstransient expression on endothelial cells in response to IL-1 or TNF(Bevilacqua et al., Science (1989) 243:1160). The time course of thisinduced expression (2-8 h) suggests a role for this receptor in initialneutrophil extravasation in response to infection and injury. Indeed,Gundel et al. have shown that antibody to ELAM-1 blocks the influx ofneutrophils in a primate model of asthma and thus is beneficial forpreventing airway obstruction resulting from the inflammatory response.(Gundel R. H. et al., J. Clin. Invest. (1991) 88:1407).

Several different groups have published papers regarding ELAM-1 ligands.Lowe et al., Cell (1990) 63:475 demonstrated a positive correlationbetween ELAM-1 dependent adhesion of HL-60 cell variants and transfectedcell lines, with their expression of the sialyl Lewis x (sLex)oligosaccharide, Neu NAc α2-3Gal-β1-4(Fucα1-3)-GlcNAc. By transfectingcells with plasmids containing an α(1,3/1,4) fucosyltransferase, theywere able to convert non-myeloid COS or CHO lines into sLex-positivecells that bind in an ELAM-1 dependent manner. Walz et al., (1990) wereable to inhibit the binding of a ELAM-1-IgG chimera to HL-60 cells witha monoclonal antibody directed against sLex or by glycoproteins with thesLex structure, but could not demonstrate inhibition with CD65 or CD15antibodies. Both groups concluded that the sLex structure is the ligandfor ELAM-1.

Information regarding the DNA sequences encoding endothelialcell-leukocyte adhesion molecules are disclosed in PCT publishedapplication WO90/13300 published Nov. 15, 1990 incorporated herein byreference. The PCT publication cites numerous articles which may berelated to endothelial cell-leukocyte adhesion molecules. The PCTpublication claims methods of identifying ELAM-ligands, as well asmethods of inhibiting adhesion between leukocytes and endothelial cellsusing such ligands and specifically refers to MILAs which are describedas molecules involved in leukocyte adhesion to endothelial cells.

As alluded to above, the ligand for ELAM, sLewis^(x), consists of atleast. sialic acid, fucose, and lactose. Lactose consists of galactoseand glucose. Sialic acid and fucose are bound to the galactose andglucose moieties of lactose, respectively. Ligands that bind to theother selectins share similar structural features. Considering theobvious medical importance of selectin ligands, significant effort hasbeen, and continues to be expended to identify the criticalphysical/chemical parameters associated with selectin ligands thatenhance, or that are required for their activity. In no small part thiseffort is being driven by the need to have selectin ligands that areinexpensive to produce. It is generally thought that it will becommercially prohibitively expensive to produce naturally occurringsLewis^(x) by either enzymatic or chemical synthesis because of thenumber of sophisticated reactions involved.

SUMMARY OF THE INVENTION

A first object of the invention is the description of medicaments thatare selectin ligands that bind to certain selectins, and that are costeffective to synthesize.

A second object of the invention is the description of medicaments thatare selectin ligands that bind to certain selectins wherein the ligandslack the lactose core structure of sLewis^(x), and have substituted inits place a spacer moiety. Relative to sLewis^(x), such medicaments arecost effective to synthesize.

A third object of the invention is a description of certain novelmedicaments that incorporate newly discovered physical/chemicalproperties associated with sLewis^(x) such that the medicaments have athree-dimensionally stable configuration for the presentation of thefunctional groups, sialic acid and fucose, of sLewis^(x), thatfacilitates binding between those groups and receptors on the selectins.Such invention compounds are represented by the following generalstructural formula I: ##STR2## wherein m and n are independently aninteger of from 1 to 5, Y and Z are independently a connecting moietyselected from the group consisting of --CH₂ --, --O--, --S--, --NR' and--NR'R"--(wherein R' and R" are independently H or an alkyl containing 1to 4 carbon atoms); X is a connecting moiety which is selected from thegroup consisting of --O--, --S-- and --N--; and --R'" may be --R" or anymoiety which does not interfere with the three-dimensional configurationof A or B so as to interfere with selectin binding and is preferably amoiety selected from the group consisting of --OR", --SR", --I, --N₃,and --NR'R", and A and B are the moieties having the saccharides shownin the boxes or analogues or derivatives thereof.

A fourth object of the invention is to provide a composition comprisingselectin ligand medicaments bound to a detectable label and/or bound toa pharmaceutically active drug such as an anti-inflammatory drug.

A fifth object of the invention is to provide a pharmaceuticalformulation containing selectin ligand medicaments which is useful intreating certain diseases.

A sixth object of the invention is to provide a description of methodsto treat or diagnose disease.

A seventh object of the invention is to provide compositions and methodsto determine the site of inflammation by administering labeledformulations of the type referred to above.

Another object of the invention is that the ligands can be labeled andthe labeled ligands used in an assay to detect the presence of selectinsin a sample.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the synthesis, structure, formulation and usageas more fully set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention may be better understood and its numerous objects,advantages and features will become apparent to those skilled in the artby reference to the accompanying drawings as follows:

FIG. 1 is a cross-sectional schematic view showing the interactionbetween white blood cells and activated endothelial cells.

FIG. 2 is a cross-sectional schematic view showing how compounds of theinvention would be used as pharmaceuticals to block ELAM-1.

FIG. 3 is a graph showing the column chromatograph of two specificcompounds; GM 1222 and GM 1279.

FIG. 4 is a graph showing the results of Elisa assays carried out todetermine the ability of two different compounds to block the binding of2,3,sialyl-Le^(x) o E-selectin IgG chimera. The compounds tested were GM1279 and GM 1398. The compounds were tested at several concentrations asshown in the figure, and the results expressed as the per cent ofcontrol binding.

FIG. 5 is a graph showing the results of Elisa assays carried out todetermine the ability of three different compounds to block the bindingof 2,3,sialyl-Le^(x) to L-selectin IgG chimera. The compounds testedwere GM 1222, GM 1279 and GM 1398. The compounds were tested at severalconcentrations as shown in the figure. The results are expressed as percent of control binding.

FIG. 6 is a graph showing the results of Elisa assays carried out todetermine the ability of GM 1221 to block the binding of2,3,sialyl-Le^(x) to L-selectin IgG chimera. The results are expressedas per cent of control binding.

FIG. 7 shows the effects of two compounds, GM 1221 and GM 1398 onthioglycollate induced peritonitis. Mice were injected with saline alone(sal/--), thioglycollate and phosphate buffered saline (TG/PBS) orthioglycollate and GM 1221 or GM 1398 (TG/GM 1221 or TG/1398), and thenumber of neutrophils in the peritoneal cavity measured.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the description of the invention reference is made to certainpublications including scientific articles and patents or patentapplications. It is the intent that each of these publications beincorporated by reference when referred to in the specification.

Before the present invention compounds and compositions containing suchand processes for isolating and using such are described, it is to beunderstood that this invention is not limited to the particularcompositions, methods or processes described as such compositions andmethods may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting since the scope ofthe present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms "a", "an" and "the" include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to "an ELAM-1"includes reference to mixtures of such molecules, reference to "theformulation" or "the method" includes one or more formulations, methodsand/or steps of the type described herein and/or which will becomeapparent to those persons skilled in the art upon reading thisdisclosure and so forth.

Some standard abbreviations used in connection with the presentinvention include: BSA, bovine serum albumin; DEAE, diethylaminoethyl;DMSO, dimethyl-sulfoxide; ELAM-1, endothelial/leukocyte adhesionmolecule-1; HPTLC, high performance thin layer chromatography; LECAM-1,leukocyte/endothelial cell adhesion molecule-1; MOPS, 3-N-Morpholino!propanesulfonic acid; NANA, N-acetylneuraminic acid; PVC,polyvinylchloride; TLC, thin layer chromatography; TFA, trifluoroaceticacid; Tris, tris (hydroxymethyl) aminomethane.

DEVELOPMENT OF THE INVENTION

It is worth noting that while the invention compounds were selected fortheir capacity to bind to certain selectins, and that therefore thisproperty is indicative of their medical activity, it cannot be excludedthat they are also exerting their favorable medical effects, either inparallel or in tandem, through additional mechanisms of action. Thus,the skilled practitioner of this art will appreciate that a key aspectof the subject invention is the description of novel medicaments, andthat Applicants intend not to be bound by a particular mechanism ofaction that may account for their prophylactic or therapeutic effects.

ELAM-1 has a lectin like domain that recognizes Sialyl Lewis(SLe^(x))tetrasaccharide as shown below in structural formula II.##STR3## The ability of SLe^(x) to bind ELAM-1 is described by Lowe etal., Cell (1990) 63:475; Phillips et al., Science (1990) 250:1130; Walzet al., Science (1990) 250:1132; and Tyrrell et al., Proc. Natl. Acad.Sci. USA (1991) 88:10372.

It has also been shown (Berg et al., J Biol Chem (1991) 265:14869; Handaet al., Biochem Biophys Res Commun (1991) 181:1223) that both ELAM-1 andGMP-140 recognize the isomeric tetrasaccharide SLe^(a) shown below asstructural formula III. ##STR4##

A key step in making the invention compounds was the realization thatboth SLe^(x) and SLe^(a) share a structural similarity in their threedimensional arrangements. Specifically, we observed that sialic acid andfucose, two functional epitopes in these tetrasaccharides, arejuxtaposed in space in a way suitable for recognition by the selectins.Most importantly, for both tetrasaccharides we identified 4 to 12 atomsassociated with the lactose core of the tetrasaccharides thatfunctionally separate sialic acid from fucose. The 4 to 12 atomsexcludes those defined as "Y" and "Z" in formula 1.

We postulated that replacement of these atoms would lead to compounds,such as those described and claimed herein, that maintain their selectinbinding activity. While 4 to 12 is the preferred number of atoms, mostpreferred is 6 to 8 atoms as denoted, shown in the figure below by Romannumerals.

For instance, a close structural examination of SLe^(x) (shown in I) ora modification thereof wherein R═OH (SLe^(x) Glc) indicates that theepitopes i.e., α-Neu5Ac and L-Fucose, are linked through six atoms (Nos.1-6) or eight atoms (Nos. i-viii) as shown in structural formula II(a)below. ##STR5## wherein R is --NHAc or --OH.

Based on this discovery, we deduced that the corresponding epitopes onthe lectin domain of ELAM-1, and the other selectins, are spaced in asimilar three-dimensional configuration such that maintenance of the 6to 8 atoms in the ligand structure would yield active ligands that aremarkedly different in structure from the naturally occurring ligand.

Using these insights, we then designed certain selectin ligands. Thismay be done by attaching sialic acid and L-fucose as such, or analogs orderivatives thereof, through 4 to 12 atoms, or presently through six oreight atoms to provide a series of compounds shown as structural formulaI. This series of compounds is designed to competitively inhibitselectins from binding to their natural ligands. These compounds can becombined with pharmaceutically acceptable excipiehts to providepharmaceutical compositions useful in a wide range of treatments.

The structures that contain the appropriate reactive functions can bereacted with suitably protected hydrophobic carriers like ceramide or aceramide mimic, steroids, diglycerides or phospholipids to formmolecules that act as immunomodulators.

The compounds can act as antagonist ligand molecules, i.e. biochemicalblocking agents by binding to selectins and preventing circulatingneutrophils from binding to endothelial cells, thereby preventing aprimary event involved in certain diseases, including the inflammatoryresponse. Agonist ligands have the opposite effect.

The compounds of the present invention are designed to provide athree-dimensionally stable configuration for functional groups on sialicacid and fucose moieties so as to allow for binding between those groupsand receptors on natural selectins. The compounds are represented by thefollowing general structural formula I(a): ##STR6## wherein m and n areindependently an integer of from 1 to 5, Y and Z are independently aconnecting moiety selected from the group consisting of --CH₂ --, --O--,--S--, --NR' and --NR'R"-- (wherein R' and R" are independently H or analkyl containing 1 to 4 carbon atoms); X is a connecting moiety which isselected from the group consisting of --O--, --S-- and --N--; and --R'"may be --R" or any moiety which does not interfere with thethree-dimensional configuration of A or B so as to interfere withselectin binding and is preferably a moiety selected from the groupconsisting of --OR", --SR", --I, --N₃, and --NR'R", and A and B are asshown in structures IV and V respectively or analogues or derivativesthereof. ##STR7##

The compounds of general structural formula I(a) can be bound to knowndrugs, for example anti-inflammatory drugs so as to targei thedrug-selectin ligand complex to a particular site of disease.Additionally, they can be formulated to provide compositions useful inassaying a sample for the presence of selectins such as ELAM-1 and/orLECAM-1, or to detect the site of inflammation in a patient, or to treatacute inflammation (or treating the inflammatory symptoms of certaindiseases) or other diseases involving the interaction of selectins onappropriate cell types.

In the general structural formula I(a) R'" has been generally defined tobe any moiety which, when attached at the R'" position, will notinterfere with the ability of the molecule as a whole to bind to aselectin receptor. More specifically, R'" will be an organic compoundwhich contains hydrogen and carbon atoms alone or in combination with O,N and/or P. Specific examples of R'" include: ##STR8## wherein R1 and R2are independently an alkyl, or alkenyl, preferably an alkyl containing1-5 carbons or containing 13 to 15 carbons; ##STR9## wherein n and m areeach independently an integer of from 15 to 24 and wherein the alkylgroup may be saturated or unsaturated; ##STR10## wherein R is--CO(CH₂)₁₄ CH₃ ; ##STR11##

The preferred example of A and B are shown, respectively, in formula IVand V. Other examples of A include α- or β- or other analogues orderivatives of sialic acid other than the N-acetyl neuraminic acidresidue shown in formula IV, Kemp's acid, glycefic acid, lactic acid,acetic acid and --SO₃ and --PO₃. The synthesis of certain analogues ofsialic acid is described in U.S. Pat. No. 5,138,044.

Preferred forms of B are the α and β forms of L-Fucose as shown informula V. The moiety B also includes substituted forms of the followingfucose structure V(a): ##STR12## wherein Me is a methyl group, R1, R2and R3 are each independently --OH, --F, --NR"R'" (wherein R" and R'"are independently alkyls containing 1 to 5 carbon atoms). Other moietiesfor B include modified fucosides such as corresponding carboxylicanalogues of fucose; inositol; substituted inositol; benzimidazole;substituted benzimidazole; guanidine; substituted butane, whereinsubstituents include --CH₂, --CHR1, --CHR2, CH₂ R3 and R1, R2, and R3are independently OH, F or NR"R'"; pentaerythritol and substitutedpentaerythritol.

Assaying Compounds of Formula I(a)

The compounds of formula I(a) can be tested for their ability to bind toa selectin receptor and/or block the binding site of the receptor andthereby prevent a natural ligand from binding to the selectin receptor.A generalized procedure for testing the compounds of formula I(a) isgiven below.

An ELISA assay is preferably used that employs recombinant fusionproteins composed of extracellular portions of the human selectinsjoined to human immunoglobulin heavy chain CH3, CH2, and hinge regions.See, for example, Walz et al., Science (1990) 250:1132; Aruffo et al.,Cell (1991) 67:35; Aruffo et al., Proc. Natl. Acad. Sci. USA. (1992)89:2292. The assay is well known in the art, and generally consists ofthe following three steps:

I. 2,3sLex glycolipid (25 picomole/well) is transferred into microtiterwells as solutions and then evaporated off. Excess, which remainedunattached, is washed off with water. The wells are then blocked with 5%BSA at room temperature for an hour and then washed with PBS containing1 mM calcium.

II. Preparation of "multivalent" receptor of the Selectin-IgG chimera iscarried out by combining the respective chimera 1 ug/ml) with biotinlabelled goat F(ab')₂ anti-human IgG (Fc specific) andstreptavidin-alkaline phosphatase diluted 1:1000 in 1% BSA-PBS (1 mMcalcium) and incubating at 37° C. for 15 min. This allows the solublemultivalent receptor complex to form.

III. Potential inhibitors such as compound of formula I(a) are allowedto react with the soluble receptor at 37° C. for 45 min. This testassumes that optimal binding, between the soluble phase receptor complexand the inhibitor (non natural ligand), occurs within this time frame.This solution is then placed in the microtiter wells that were preparedin step I. The plate was incubated at 37° C. for 45 minutes to allow thesoluble receptor to bind to its natural iigand. In the presence of astrong inhibitor only a few receptors should be free to bind to themicrotiter plate coated with the natural ligand.

The positive control is the signal produced by the soluble receptor whenit is allowed to react with 2,3sLex glycolipid in the microtiter wellsin the absence of any inhibitor. This is considered 100% binding. Thesignal produced by the receptor that has been previously treated with aninhibitor (recorded as O.D.), is divided by the signal produced by thepositive control and multiplied by 100 to calculate the % receptor boundto the well in the presence of the inhibitor. The reciprocal of this isthe % inhibition.

Referring now to FIG. 1, a cross-sectional view of a blood vessel 1 isshown. The vessel wall 2 is lined intemally with endothelial cells 3.The endothelial cells 3 can be activated causing the cells 3 tosynthesize ELAM-1 which is displayed in FIG. 1 as a triangular surfacereceptor 4. Both red blood cells 5 and white blood cells (6A, 6B) flowin the vessel 1. The white blood cells display carbohydrate ligands 7which have chemical and physical characteristics which allow the ligands7 to bind to the receptors 4. Once the ligand 7 binds to the receptor 4,the white blood cell is brought through the vessel wall 2 as is shownwith the white blood cell 6A. The white blood cells 6B brought into thesurrounding tissue 8 can have positive effects, such as fightinginfection, and negative effects, such as inflammation.

An important aspect of the present invention can be described byreferring to FIG. 2. The compounds of a formula I(a) are shown as 7A andcan adhere to a selectin such as ELAM-1 by themselves and can beformulated into pharmaceutical compositions, which when administeredwill effectively block the ELAM-1 and prevent the adhesion of a ligand 7connected to a white blood cell 6. By administering pharmaceuticallyeffective amounts of the compounds 7A, some, but not all, of the whiteblood cells will not reach the surrounding tissue. By slowing the rateat which the white blood cells reach the surrounding tissue,inflammation can be prevented and/or alleviated.

It is known that for an acute inflammatory response to occur,circulating neutrophils must bind to and penetrate the vascular wall andaccess the site of injury. Several molecules have been implicated inthis interaction, including a family of putative carbohydrate ligandsand their receptors. One molecule that was previously identified is theendogenous carbohydrate ligand for endothelial leukocyte adhesionmolecule-1 (hereinafter ELAM-1). The present invention provides a familyof molecules which bind as the endogenous ligands and thereby blockother selectin receptors such as LECAM-1 receptors.

The compounds of Formula I(a) may also be labeled using standardradioactive, fluorescent, enzymic or other label for analytical ordiagnostic purposes. In general, the significant portion of thecompounds of Formula I(a) is the compound shown; the embodiments of thesubstituent R'" will depend on the intended use. Suitable embodimentsfor this substituent will be apparent to those of skill in the art.

Preferred embodiments of the ligands of the invention are those whereinthe substituent represented by A or B is an N-acetylneuramyl residue andB is fucose.

In order for a ligand of the invention to bind to a selectin receptorsuch as an ELAM-1 receptor the ligand need not include the identicalatoms in the identical configuration as per structural formula I(a) butmust have (1) a stable three dimensional conformation as shown informula I(a) or (2) a substantially equivalent configuration to thatshown in formula I(a). The equivalency of any other ligand will relateto its physical three dimension structure and the electron configurationof the molecule and in particular the charge related characteristicspresented by the groups present on the A and B moieties shown informulae IV and V. In order for a molecule of the invention to have asubstantially equivalent structure to that shown in formula I(a) themolecule must bind to a selectin receptor (e.g. an ELAM-1 receptor) tothe same degree or greater as the compound of formula I(a) when themolecule is allowed to bind to the receptor under physiologicalconditions.

Assay to Identify Ligand (General)

Candidate ligands can be assayed for their ability to adhere to ELAM-1.The method comprises attaching candidate ligands of formula I(a) to asubstrate surface and then contacting the substrate surface thereon withrecombinant cells, that are genetically engineered to express highlevels of ELAM-1, for a sufficient time to allow the cells to adhere tothe substrate bearing the candidate ligand. Thereafter, centrifugalforce or other appropriate methodology is applied so as to separate awaythe cells which do not adhere to the substrate. Candidate ligands whichadhere to ELAM-1 are determined via the labels on the cells. Suchmolecules are isolated, characterized, and their structure specificallyidentified.

Radiolabeled COS cells expressing cell surface ELAM-1 can be used asprobes to screen compounds of the invention. ELAM-1 transfected COScells will adhere to a subset of compounds of the invention which can beresolved on TLC plates or adsorbed on PVC microtiter wells. Adhesiontests are preferably done under physiological conditions. Adhesion tothese compounds may require calcium, but will not be inhibited byheparin, chondroitin sulfate, keratin sulfate, or yeast phosphomannan(PPME). Monosaccharide composition, linkage analysis, and FAB massspectrometry of the purified compounds will indicate that the ligandsfor ELAM-1 share common structural characteristics which generallyrelate to the moieties A and B and the position in which they are held.

One mechanism by which the compounds of the invention could mediateintercellular events would involve the recognition of these compounds onone cell (e.g., an endothelial cell) by a specific carbohydrate-bindingprotein (lectin) on an opposing cell (e.g., a leukocyte). Data generatedin connection with the present invention indicate that acidicglycolipids isolated from leukocytes and ELAM-1 function as such anoligosaccharide-lectin pair, participating in the interaction ofneutrophils with the surface of cells of activated vascular endothelium.Many protein-protein interactions have been implicated inneutrophil-endothelium transmigration (see Lo et al., J. Immunol. (1989)143:3325; Osborn et al., Cell (1989) 59:1203; Larsen et al., Cell (1989)59:305; and Arnaout Blood (1990) 75:1037). The present inventors believeit is likely that this lectin-carbohydrate interaction is only one stepin a series that result in neutrophil extravasation. The adhesion ofELAM-1 for compounds of the invention has been tested. Accordingly, suchcompounds are believed to be useful in mediating a specific, butpossibly weak adhesion that is then stabilized and elaborated by theparticipation of other receptors. Compounds with the structural andfunctional characteristics described herein, or modifications of thesestructures, are believed to be capable of blocking the interaction ofneutrophils with activated vascular endothelium mediated by ELAM-1, andhence provide useful pharmaceutically active agents which can interruptthe adverse effects involved in the interaction of ELAM-1 andcirculating neutrophils, e.g., prevent or reduce inflammation.

Identification of Compounds Which Act as ELAM-1 Ligands UsingRecombinantly Produced Receptor

A complete cDNA for the ELAM-1 receptor was obtained by PCR startingwith total RNA isolated from IL-1 stimulated human umbilical veinendothelium. The resulting cDNA was inserted into the CDM8 plasmid (seeAruffo et al., Proc Natl Acad Sci USA (1987) 84:8573) and the plasmidamplified in E coli. Plasmid DNA from individual colonies was isolatedand used to transfect COS cells. Positive plasmids were selected bytheir ability to generate COS cells that support HL-60 cell adhesion.DNA sequencing positively identified one of these clones as encoding forELAM-1 (Bevilacqua et al., Science, (1989) 243:1160; Polte et al.,Nucleic Acids Res (1990) 18:1083; Hession et al., Proc Natl Acad Sci USA(1990)87:1673). These publications are incorporated herein by referencefor their disclosure of ELAM-1 and genetic material coding for itsproduction. The complete nucleotide sequence of the ELAM-1 cDNA andpredicted amino acid sequence of the ELAM-1 protein are given in theabove cited article by Bevilacqua et al., which DNA and amino acidsequences are incorporated herein by reference (see also published PCTpatent application WO90/13300 which was published Nov. 15, 1990, whichis incorporated herein by reference).

COS cells, expressing membrane-bound ELAM-1, were metabolicallyradiolabeled with ³² PO₄. These labeled cells can be used as probes intwo assay systems to screen for recognition of the compounds of formulaI(a). More specifically, compounds of formula I(a) may be adsorbed tothe bottoms of PVC microtiter wells or resolved on TLC plates. In eitherassay the compounds may be probed for their ability to support adhesionof ELAM-transfected COS cells, untransfected COS cells, or COS cellstransfected with a plasmid containing an irrelevant cDNA, underconditions of controlled detachment force (see Swank-Hill et al., AnalBiochem (1987) 183:27; and Blackburn et al., J. Biol Chem. (1986)261:2873 each of which is incorporated herein by reference to disclosethe details of such assaying methodology).

It has been indicated that R'" of structural formula 1(a) may be alinker which may be any suitable and attachable moiety including aceramide or a protein or peptide and is preferably a group with areactive group thereon which allows it to covalently bind to a substrateor pharmaceutically active drug. In one embodiment of the invention the"linker" connects one or more ligands to a support base. The supportbase is then contacted with a sample to assay for the presence of adesired selectin in the sample.

The "linker" can be used to attach a pharmaceutically effective drug tothe compound at the R' position. The (Ligand-Linker-Drug) conjugate thusformed provides an effective drug delivery system for the linked drug. Amethod of attaching any moiety at the R' position is shown in ReactionScheme V.

NSAID or non-steroidal, anti-inflammatory drugs such as naproxen oribuprofen which act as anti-inflammatory agents could be administeredbound to the modified ligand and could be administered systemically insmaller amounts than usual while obtaining an equivalent effect or evengreater anti-inflammatory effect at the site of inflammation. Any otherdrugs which might be attached include, but are not limited to,antibiotics, vasodilators and analgesics. Such a drug delivery systemwould reduce any systemic effect normally caused by the drug in that thedrugs could be administered in amounts of one-half to one-tenth thenormal dose and still obtain the same anti-inflammatory result at thesite of inflammation.

Method of Synthesis (General)

The compound of formula I(a) can be made using the general and specificsynthesis schemes and examples described below. However, those skilledin the art will recognize variations thereof which are intended to beencompassed by the present invention. In general, the A and B moietiesof formula I(a) must be connected and held in a desiredthree-dimensional configuration. A simple reaction scheme foraccomplishing such is shown below: ##STR13## wherein each X isindependently selected from the group consisting of --OH, --NH₂, --SH,and halogens such as Cl and Br; A and B are, respectively, a sialic acidand L-Fucose and their respective bioisosteres. A more specific versionof reaction Scheme I is shown below: ##STR14##

A reaction scheme wherein the fucose is attached is shown below.##STR15##

Another version of a reaction scheme wherein A and B are in the desiredconfiguration is given below: ##STR16##

It is possible to alter a compound of formula I(a) to obtain a compoundwhich is labeled or attached to any other desired compound.

A general reaction scheme for obtaining such is as follows: ##STR17##The compound shown above can then be reacted with a fluorescent probe, amultivalent compound, a ceramide, cholesterol or other lipid components,or a pharmaceutically active drug such as an anti-inflammatory drug.

Use and Administration

The compounds of the invention such as various ligands of structuralFormula I(a) can be administered to a subject in need thereof to treatthe subject by either prophylactically preventing inflammation orrelieving it after it has begun. The ligands are preferably administeredwith a pharmaceutically acceptable carrier, the nature of the carrierdiffering with the mode of administration, for example, oraladministration, usually using a solid carrier and I.V. administration aliquid salt solution carrier. The formulation of choice can beaccomplished using a variety. of excipients including, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin cellulose, magnesium carbonate, and the like. Oralcompositions may be taken in the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders.Particularly useful is the administration of the compounds directly intransdermal formulations with permeation enhancers such as DMSO. Othertopical formulations can be administered to treat dermal inflammation.

A sufficient amount of compounds would be administered to bind to asubstantial portion of the selectin expected to cause or actuallycausing the disease, for example, inflammation so that inflammation caneither be prevented or ameliorated. Thus, "treating" as used hereinshall mean preventing or ameliorating the appropriate disease.Typically, the compositions of the instant invention will contain fromless than 1% to about 95% of the active ingredient, preferably about 10%to about 50%. Preferably, between about 10 mg and 50 mg will beadministered to a child and between about 50 mg and 1000 mg will beadministered to an adult. The frequency of administration will bedetermined by the care given based on patient responsiveness. Othereffective dosages can be readily determined by one of ordinary skill inthe art through routine trials establishing dose response curves.

When determining the dose of compounds to be administered which blockselectin receptors, it must be kept in mind that one may not wish tocompletely block all of the receptors. In order for a normal healingprocess to proceed, at least some of the white blood cells orneutrophils must be brought into the tissue in the areas where thewound, infection or disease state is occurring. The amount of theligands administered as blocking agents must be adjusted carefully basedon the particular needs of the patient while taking into consideration avariety of factors such as the type of disease that is being treated.

It is believed that the compounds of the present invention can be usedto treat a wide range of diseases, including diseases such as rheumatoidarthritis and multiple sclerosis. The compositions of the inventionshould be applicable to treat any disease state wherein the immunesystem turns against the body causing the white ceils to accumulate inthe tissues to the extent that they cause tissue damage, swelling,inflammation and/or pain. The inflammation of rheumatoid arthritis, forexample, is created when large numbers of white blood cells quicklyenter the joints in the area of disease and attack the surroundingtissues.

Formulations of the present invention might also be administered toprevent the undesirable aftereffects of tissue damage resulting fromheart attacks. When a heart attack occurs and the patient has beenrevived, such as by the application of anticoagulants or thrombolytic(e.g., tPA), the endothelial lining where a clot was formed has oftensuffered damage. When. the antithrombotic has removed the clot, thedamaged tissue beneath the clot and other damaged tissue in theendothelial lining which has been deprived of oxygen become activated.The activated endothelial cells then synthesize the ELAM-1 receptorswithin hours of the cells being damaged. The receptors are extended intothe blood vessels where they adhere to glycolipid ligand molecules onthe surface of white blood cells. Large numbers of white blood cells arequickly captured and brought into the tissue surrounding the area ofactivated endothelial cells, resulting in inflammation, swelling andnecrosis which thereby decreases the likelihood of survival of thepatient.

In addition to treating patients suffering from the trauma resultingfrom heart attack, patients suffering from actual physical trauma couldbe treated with formulations of the invention in order to relieve theamount of inflammation and swelling which normally result after an areaof the body is subjected to severe trauma. Other disease states whichmight be treatable using formulations of the invention include varioustypes of arthritis and adult respiratory distress syndrome. Afterreading the present disclosure, those skilled in the art will recognizeother disease states and/or symptoms which might be treated and/ormitigated by the administration of formulations of the presentinvention.

Other modes of administration will also find use with the subjectinvention. For instance, the ligand molecules of the invention can beformulated in suppositories and, in some cases, aerosol and intranasalcompositions. For suppositories, the vehicle composition will includetraditional binders and carriers such as, polyalkylene glycols, ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),preferably about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

The compounds of the instant invention may also be administered asinjectables. Typically, injectable compositions are prepared as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection may also be prepared.The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles. Compounds of Formula I can be mixedwith compatible, pharmaceutically acceptable excipients.

Suitable vehicles are, for example, water, saline, dextrose, glycerol,ethanol, or the like, and combinations thereof. In additipn, if desired,the vehicle may contain minor amounts of auxiliary substances such aswetting or emulsifying agents or pH buffering agents. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in the art. See, e.g. Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 17th edition, 1985. The composition orformulation to be administered will, in any event, contain a quantity ofthe compounds adequate to achieve the desired state in the subject beingtreated.

The various compounds of the present invention can be used by themselvesor in combination with pharmaceutically acceptable excipient materialsas described above. However, the compounds of the invention can be madeas conjugates wherein they are linked in some manner (e.g., via the R'moiety) to a label. By forming such conjugates, the compounds can act asbiochemical delivery systems for the label so that a site of disease canbe detected.

For instance, carbohydrates can be labelled by a variety of procedures,for example: esterification of hydroxyl bonds to form a structurecapable of complexing directly with a radioisotope or nmr enhancer;reaction of the carbohydrate with amino diacetic acid (IDA) in organicsolvent to form an N-linked glycoside derivative which would be capableof complexing with a radioisotope via the nitrogen and oxygen atoms ofthe IDA group; or coupling of the carbohydrate to amino acids which maybe labelled directly (e.g. cysteine, tyrosine) or labelled via abifunctional chelating agent (e.g., lysine). Appropriate radioactiveatoms would include, for example, technetium 99m (^(99m) Tc), iodine-123(¹²³ I) or indium-111 (¹¹¹ In) for scintigraphic studies, or for nuclearmagnetic resonance (nmr) imaging (also known as magnetic resonanceimaging, mri), a label such as gadolinium, manganese or iron, or apositron-emitting isotope such as iodine-124, fluorine-19, carbon-13,nitrogen-15 or oxygen-17.

The compounds of the invention may be prepared in a sterile,non-pyrogenic medium and injected into the bloodstream of a patient at adose to be determined in the.usual wayby-the physician or radiologist.After a sufficient period for a good balance to have been reachedbetween (i) specificity of binding to activated endothelium compared tonon-specific distribution and (ii) total amount of compound on activatedendothelium, the compound is imaged in a conventional way, according tothe nature of the label used.

The compounds of the invention could also be used as laboratory probesto test for the presence of a selectin receptor such as a receptor ofELAM-1 in a sample. Such probes are preferably labeled such as with aradioactive label. There are a number of known labels includingradioactive labeled atoms, e.g. radioactive C, O, N, P, or S,fluorescent dyes and enzyme labels which can be attached to compounds ofthe invention using known procedures. Labels as well as methods ofattaching labels to sugar moieties are disclosed in U.S. Pat. No.4,849,513 issued Jul. 18, 1989 to Smith et al. which patent isincorporated herein by reference to disclose labels and methods ofattaching labels.

EXAMPLES

The following examples are provided so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake compounds and compositions of the invention and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to insure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.) but some experimental errors and deviationshould be accounted for. Unless indicated otherwise, parts are parts byweight, temperature is in degrees C., and pressure is at or nearatmospheric.

It is important to note that the invention compounds described belowhave 4 to 12 atoms associated with the lactose core of thetetrasaccharides that separate sialic acid from fucose, or that separateanalogues or derivatives of sialic acid from fucose and vice versa.

Example 1 1-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-Hexane-6-ol (3)

Hexane-1,6-diol (6 g. 25 mole was dissolved in 2:1 1,2 dichloroethane-N,N-dimethyformanide (30 mL), Bu₄ NBr (4.0 g,) was added and the mixturewas stirred at R. T. (3h) under inert (argon) atmosphere. A solution ofmethyl 2,3,4-tri-O -benzyl-1-L-fucopyranosyl bromide prepared by thereaction of 2,3,4-tri-O-benzyl-thio-α-L-fucopyranoside (1 g) withbromine (70 μl)! in dichloroethane (1 mL) was added into the reactionmixture. T.L.C. (5:1:0.5 Toluene-Acetone-MEOH) showed formation ofproduct. Multiple elution on T.L.C. (6:1×1 and 8:1×2Toluene-Ethylacetate) indicated one major (RF=0.34) and one minor(Rf=0.30) product. Filtration, evaporation and column chromatographicpurification afforded the title compound 3 (major product) as a syrup(730 mg), α!²² _(D) -44°, α!²² ₄₃₆ -79° (c, 2.2 CHCl₃). ¹ H-NMR (CDCl₃):7.5-7.2 (m, 15H, aromatic, 5.00-4.61 (m, 7H, 3 CH₂ Ph, H-Fucp), 4.78 (d,J 3.4 Hz, H-1 fuc1), 4.03 (dd, 1H, J 3.5Hz, 1.1 Hz, H-2 Fucp), 3.93 (dd,1H, J 2.75 Hz, 10.2 Hz, H-3 Fucp), 3.86 (bq, 1H, J 6 Hz, H-5 Fucp), 3.64(bd, 1H, J 2.1 Hz, H-4 Fucp) 3.58 and 3.42 (2m, 2H --CH₂) and 1.1 (d,3H, J 6.51Hz, CH₂ OH), 3.54 (t, 2H, J 6.6 Hz, --OCH₂ --), 20 (bs, 2H,--OH), 1.62 and 1.52 (2m, 4H 2CH₂), 1.35 (m, 2CH₂) and 1.1 (d, 3H, J6.51Hz, CH₃ Fucp). ¹³ C-NMR (CDCl₃): δ138.9, 138.5 (3 C-1 Ph), 9.73(C-1), 79.3, 77.7, 76.43 (C-2), 66.07 (C-5), 7-4.7, 73.18, 73.15 (3 CH₂Ph), 67.99, 62.56 (2 OCH₂) 32.5, 29.3, 25.97, 25.48 (4 CH₂) and 16.6(CH₃). Calcd, for C₃₃ HA₂ O₆, Exact Mass. 535.29. Found by f.a.b.-m.s.:535.6 (M+1)⁺, 580.0 (M+NO₂)⁻, 687.8 (M+NBA)⁻.

Further elution afforded the mixture of α, and β fucosides (65 mg)followed by the pure β-anomer, (140 mg), α!²² _(D) -0.85, α!²² 436 ⁻¹.8(c 1.77, CHCl₃); ¹ H-NMR (CDCL₃): δ4.3 (d, 1H, J 7.7Hz, H-1), 3.8 (dd,1H, J 7.7Hz); 1.17 (d, 3H, J 6.4Hz, CH₃ ¹³ C-NMR (CDCL₃); δ103.8(C-1),82.5, 79.4 (C-3), 76.2 (C-2), 70.2 (C-5, moved downfield) 75.1 (C-4,moved downfield), 74.5, 73.1, 69.6, 62.8, 32.6, 29.7, 25.9, 25.5 (6CH₂), 16.86 (CH₃). Total yield 935 mg (81%).

Example 2 Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-2-O-6-O-(2,3,4-tri-O-benzyl -α-L-fucopyranosyl)Hexyl-D-glycero-α-D-galacto-2-nonulopyranosonate(5)

Compound 3 (329 mg) and 4(970 mg) were dissolved in dry propionitrile(5.0 mL), molecular sieve 4A° (1 g) was added and the mixture wasstirred for 2h at R. T. Silver Triflate (1.1 g) was added and thesepturn sealed flask, containing the mixture was cooled to -73° C. Asolution of methyl sulfenyl bromide (950 μl) in dichloroethane (989.9mg/2.55 mL was injected into the reaction mixture.

After 2h, the reaction was terminated by the addition of a solution ofEt₃ N in CHCl₃ (1 mL in 5 mL), filtration, washing with water, drying(MgSO₄), filtration and evaporation of the organic layer afforded crudeproduct mixture which was column chromatographically purified to affordthe name compound 5 (178 mg, 30%) as a syrup α!_(D) -24°; α!₄₃₆ -42.3°(c 1.55, CHCl₃). ¹ H-NMR (CDCl₃). δ7.4-72.5 (m, arom), 5.38 (m, H-8Neu5Ac), 5.35-531 (2d, H-7 Neu5AC), 520 (d, NH), 5.00-4.63 (bd, 3 CH₂Ph), 4.77 (d, J 3.8 Hz, H-l Fuc), 4.31 (2dd, H-9a Neu5Ac), 4.02 (2dd,H-2 Fuc), 2.75 (dd, J 467Hz, 12.82 Hz, H-3e Neu5AC), 1.56 bm, 4H, (CH₂)₂!, 1.31 bm, 4H, (CH₂)₂ !, 1.10 (d, J 6.53 Hz, CH₃ Fuc). ¹³ C-NMR(CDCl₃): δ170.019, 170.648, 170.257, 170.151, 170.060, 168.498 (6CO),138-998, 138-725, 138-604 (3 Cl-Ph), 98.72 (C2, Neu5Ac), 97.38 (C-1Fuc), 74.77, 73.25, 73.16, 67.98, 65.01, 62.34, (3--OCH₂ Ph, 2--OCH₂--CH₂, C-9 Neu5Ac), 52.63 (OCH₃), 49.34 (C-5 Neu5Ac), 38.09 (C-3Neu5Ac), 29.55, 29.36, 25.90, 25.71 (4CH₂ --), 23.18 (NCOCH₃), 21.2,20.86, 20.79 (1:2:1, OCOCH₃) and 16.65 (CH₃ Fuc).

Example 3 1-O-α-Neu5Ac-(6-O-α-L-Fucopyranosyl)-Hexane (6, GM 1398)

Compound 5 was deacetylated in methanol using sodium methoxide. Afterneutralization with cationic resin (IR120-H⁺) at ˜0°-5° C., the solutionwas filtered and evaporated to give a syrup. The syrupy material wasdissolved in 1:1 10% aqueous methanol-p-dioxane (5 mL) and reacted with0.2 M aqueous potassium hydroxide (1.5 mL) for 15 h at 0° C.--r.t. Thereaction mixture was neutralized with IR 120(H⁺), filtered andevaporated to give a syrup (R_(f) =0.37, TLC in 2:1 CHCl₃ -10% aq.MeOH). This material was dissolved in 10% aq. MeOH (5 ml) andhydrogenated at atmospheric pressure over 5% Pd-C (100 mg). TLC of thehydrogenated product showed only one product (R_(f) =0.06, 2:1 CHCl₃-10% aq. MeOH). The reaction mixture was filtered through Celite,concentrated, and the product was purified by chromatography from abiogel P2 column using water as the eluant. Appropriate fractions werepooled and lyophilized to afford pure 9.¹ H-NMR data (D₂ O-acetone):δ4.85 (d, J 3.85Hz, 1H-fuc), 402 (q, 1H, H-5 fuc), 3.85-3.35 (ringprotons and 2OCH₂), 2.7 (dd, J 4.64 Hz, H-3e Neu5Ac), 2.0(s, 3H,NHCOCH₃), 1.65-1.45 (m, 5H, H-3a Neu5Ac and 2 CH₂), 1.35 (bm, 4H, 2CH₂)and 1.18 (d, J 6.65Hz, CH₃ --Fuc). Molec. Formula, C₂₃ H₄₁ NO₁₄ (Mol.Wt., 555.58, Exact mass, 555.253). Found: 556.5 (M+1)⁺, 688.2 (M+Cs)⁺,292.2 (2,3-dehydro Neu5Ac), 265.3 (M-Neu5Ac)⁺ and 554.2(M-1)⁻,290.4(Neu5Ac)⁻.

Example 4 6-O-R,S)-1-Methoxycarbonyl-ethyl!-Hexanyl-2,3,4-Tri-0-benzyl-α-L-Fucopyranoside(8)

Compound 1 (2.9 g, 6.2 mmoL) and 2 (37 g, 313.0 mmoL) were transferredinto 500 mL flask, dried under high vacuum (8h), and dissolved in 6:1dichloromethane-N,N-dimethylformanide (350 mL). Molecular sieves 4A (5g) was added and the solution was stirred (2 h), then cooled (0° C.)before the addition of CuBr₂ (2.33 g) and Bu₄ NBr (3.7 g), into it. Thedark reaction mixture was stirred at R. T. (3-4 h), filtered throughCelite and the total flitrate was washed with saturated NaHCO₃,saturated NaCl and water. The organic layer was dried (MgSO₄), filteredand evaporated to give a syrup. Column chromatography from dry silicagel column (400 g), using 10:1 (300 mL), 40:1 (1.5L) and 20:1toluene-acetone afforded the pure product (7) as a syrup (1 g.).Molecular formula C₃₃ H₄₂ O₆ (Mol. wt. 534.70). Found: 535.6 (M+1)⁺,417.4 (M-117)⁺ and 580.0 (M+NO₂)⁻, 687.8 (M+NBA)⁻. ¹ H-and ¹³ C-NMRindicated it to be a mixture of α- and β- L-fucopyranosides.

Compound 7 (0.97 g) was dispersed in toluene (50 mL) in a 100 mL flaskequipped with Dean-Stark assembly. Toluene (15 mL) was distilled offfollowed by the addition of (Bu₃ Sn)₂ O (1.52 mL) into the reactionmixture. More toluene (20 mL) was distilled off and the resultingsolution was refluxed (2-3h). All the toluene was evaporated off on arotatory evaporator using a short neck distillation assembly fitted witha stop cock. The evacuated flask containing the stannylated material wasshut off using the stop cock and removed from the rotatory evaporator.Argon was introduced into the flask followed by methyl2-chloropropionate (1.5 mL). The content was stirred and heated(95°-100° C.) under an argon atmosphere for 12 h. The mixture wascooled, crushed ice followed by dichloromethane (15 mL) was introducedinto the flask. The total content was transferred into a separatoryfunnel, the organic layer was washed with water, separated and dried(MgSO₄). Filtration and evaporation of the dichloromethane affordedcrude product which was transferred on dry packed silica gel (150 g)column and eluted with toluene (120 mL) followed by 60:1 (200 mL) and80:1 toluene-acetone. The anomeric mixture of products (8) was isolatedas a syrup. ¹³ C-NMR (CDCl₃ --TMS):δ170.14 (CO), 103.79 (C1 β-L-FUCp,97.45 (C1 α-L-Fucp; α:β5.51), 52.8, 52.56 (OCH₃), 21.49 (Lactyl CH₃),16.6, 16.8 (CH₃ of α- and β-L-Fucp).

Example 5 1-O-(α-,β- L-Fucopyranosyl)-6-O- (R,S)-1-Carboxyethyl!-Hexane(9)

Syrupy 8 was dissolved in 10% aqueous methanol and hydrogenated (2 days)in the presence of 10% Pd-C under atmospheric pressure. Completion ofthe reaction was determined by TLC (5:1:0.2 toluene-acetone-10% aq.MeOH). The reaction mixture was filtered through Celite and the clearflitrate was evaporated to dryness. ¹ H-NMR of the product indicatedcomplete absence of the aromatic signals.

The crude product was dissolved in 1:1 p-dioxane-MeOH (2.5 mL), 10% aq.MeOH (3.0 mL) was added followed by the addition of 0.2M aq. KOH (1.5mL) at 0°-5° C. Reaction was then continued for 8-10 h at R. T. TLC(every hour in 5:1:1 toluene-acetone-10% aq. MeOH) indicated conversioninto product with lower R_(f). The reaction mixture was neutralized withIR 120 (H⁺), filtered and evaporated. The syrup was charged on drypacked silica gel column and eluted with 5:1:1 toluene-acetone-10% aq.MeOH. The GC-MS of the pure product (R_(f) =0.17), as its permethylatedderivative showed one major and one minor component (due to the α- andβ-L-Fucopyranose), having the same fragmentation pattern with major m/eat 189 (2,3,4,-Tri-O-methyl-L-Fucose)+, 157 m/e 189-32(OCH₃)!, 115 and88 i.e. total 203 due to {O-Hexyl-O-CH(COOCH₃) (CH₃)}! (FIG. 3).

Example 6 Synthesis of O-Hexamethylene linked α-, β-NeuNAc and α-,β-L-Fucopyranose (GM 1222 and GM 1279)

Compound 7 (203.6 mg) and donor 4 (597 mg) were dissolved inacetonitrile (7.0 mL) and stirred (1 h) in the presence of molecularsieves 4 (2 g). Silver triflate (764 mg) was added and the mixture wascooled (-30° C.), before the addition of a solution of methyl sulfenylbromide (355 uL of a stock solution containing 989.9 mg/2.55 mL). At theend of the reaction (2-3 h), the reaction was worked up in the usualmanner and the crude product was charged on a silica gel (350 g) columnand eluted successively with 5:1:0.1 (180 mL) and 7:1:0.1(Toluene-Acetone-MeOH). The product was isolated as mixtures in twomajor fractions (76-83) and (84-110) respectively. ¹³ C NMR of bothfractions showed: δ 171.0, 170.65, 170.25, 170.18, 170.15, 170.056 and168.5 (CO), 138.99-138.57 (6 C-1 Ph), 103.79 (β-fuc C-1), 98.71 (C-2Neu4Ac), 97.38 (α-fuc C-1), 52.63 (OCH₃), 49.36 (C-5 Neu5Ac), 38-08 (C-3Neu5Ac), 29.7-29.3 (3CH₂), 25.9-25.7 (3CH₂), 23.2 (NHCOCH₃), 21.1-20.8(OCOCH₃) and 16.88, 16.67 (CH₃ of α-, β-L-Fuc).

NMR indicated that the first product mixture contained largerproportions of α-fuc and β-NeuNAc.

Deprotection

Each pool of fractions were deprotected separately. Deacetylation wascarried out in methanol (dry) using catalytic sodium methoxide. Afterneutralization (IR 120H⁺), filtration and evaporation, the syrups weredissolved in 2:1 MeOH-P-Dioxane (3 mL) and saponified by the addition of0.2 M aqueous KOH (1.5 mL) to give products (R_(f) =0.35 and 0.33),T.L.C. solvent (2:1 CHCl₃ -10% aqueous MeOH).

Expected molecular formula: C₄₄ H₅₉ NO (M. wt: 825.96; Exact Mass:825.39). Found: 848.5 (M+Na)⁺ and 824.6 (M-1)⁻.

The syrupy products were dissolved in 10% aqueous MeOH and hydrogenatedover 10% Pd-C at 1 atmosphere until TLC (2:1 CHCl₃ -10% aqueous MeOH)indicated absence of the starting compounds and appearance of newproducts (R_(f) =0.17). Filtration, evaporation and lyophilizationsafforded two mixtures (GM 1222 and GM 1279). Expected molecular formula:C₂₃ H₄₁ NO₁₄ (M. wt. 555.552, Exact mass: 555.253). Found: 556.5 (M+1)⁺,578.5 (M+Na)⁺, 292.2 (2,3-enesialic acid+1)⁺, 265.3 (M-291)⁺ and 554.2(M-1)⁻.

Analysis and Testinq

Analysis and testing of two specific products referred to as GM 1279 andGM 1222 is described below. The structure of each of these compounds wasshown above. Comparison of the T.L.C. of α-Neuraminidase treated GM 1222and GM 1279 showed that both contained α-linked sialic acid as the majorcomponent. However, NMR had indicated that the first of the two mixturescontained larger proportion of α-L-fucose. These samples wereindividually analyzed by Dionex (Carbopak DA-1 column with isocraticrunning 0.1M NaOH) and were found to contain equal proportions of Fucoseand Neu5Ac. The elution profile in.dicated the presence of four distinctsignals for both GM 1222 and GM 1279, (FIG. 4). Based on the NMRinformation and T.L.C. data after neuraminidase treatment, the majorsignals, eluting at a slower rate, were designated to compounds havingα-linked sialic acid. The faster eluted minor components containedβ-linked L-fucoside. Pool 2 (GM 1279) contained higher proportions ofβ-L-fucopyranoside (O-linked through hexamethylene to a O-α-sialicacid).

Example 7 Selectin Binding

An ELISA assay was employed that uses recombinant fusion proteinscomposed of extracellular portions of the human selectins joined tohuman immunoglobulin heavy chain CH3, CH2, and hinge regions. See, forexample, Walz et al., Science (1990) 250:1132; Aruffo et al., Cell(1991) 67:35; Aruffo et al., Proc. Natl. Acad. Sci. USA (1992) 89:2292.The assay is well known in the art, and generally consists of thefollowing three steps:

I. 2,3sLex glycolipid (25 picomole/well) was transferred into microtiterwells as solutions and then evaporated off. Excess, which remainedunattached, was washed off with water. The wells were then blocked with5% BSA at room temperature for an hour and then washed with PBScontaining 1 mM calcium.

II. Preparation of "multivalent" receptor of the Selectin-IgG chimerawas carried out by combining the respective chimera 1 ug/mL) with biotinlabelled goat F(ab')₂ anti-human IgG (Fc specific) andstreptavidin-alkaline phosphatase diluted 1:1000 in 1% BSA-PBS (1 mMcalcium) and incubating at 37° C. for 15 min. This allowed the solublemultivalent receptor complex to form.

III. Potential inhibitors such as compound of formula I(a) were allowedto react with the soluble receptor at 37° C. for 45 min. This testassumes that optimal binding, between the soluble phase receptor complexand the inhibitor (non natural ligand), would have occurred within thistime frame. This solution was then placed in the microtiter wells thatwere prepared in step I. The plate was incubated at 37° C. for 45minuies to allow the soluble receptor to bind to its natural ligand. Inthe presence of a strong inhibitor only a few receptors should be freeto bind to the microtiter plate coated with the natural ligand.

The positive control is the signal produced by the soluble receptor whenit is allowed to react with 2,3sLex glycolipid in the microtiter wellsin the absence of any inhibitor. This was considered 100% binding. Thesignal produced by the receptor that had been previously treated with aninhibitor (recorded as O.D.), was divided by the signal produced by thepositive control and multiplied by 100 to calculate the % receptor boundto the well, or as expressed in the figures, the per cent of controlbinding. Several of the compounds described herein were tested usingthis assay.

The results shown in FIG. 4 show that both GM 1279, and GM 1398 inhibitE-selectin binding to 2,3sLex glycolipid. FIG. 5 shows that GM 1279, andGM 1398 also inhibit L-selectin binding in a concentration dependentmanner. Further, the figure also shows that GM 1222 blocks L-selectinbinding at concentrations lower than GM 1279 and GM 1398.

The structures of GM 1398, GM 1279, and GM 1222, are shown below.##STR18##

The results shown in FIG. 6 indicate that the compound GM 1221 inhibitsL-selectin binding to 2,3sLex glycolipid. Note that at all fourconcentrations of the compound tested there is significant inhibitoryactivity. The structure of GM 1221 is shown below: ##STR19##

In addition to the ligands described above, other ligands could beobtained by selecting more rigid spacers in order to maintain theappropriate statistical average distance between the sialic acid andfucose moieties in space thereby improving the inhibitory property ofsuch structures towards the selectins. Further modifications of thesecompounds e.g., attaching them through chemical linkages on appropriatemolecular supports and use of analogs or derivatives of sialic acid andL-fucose are also considered to be within the scope of the presentinvention.

Example 8 Treatment of Sepsis

A number of the complications associated with sepsis arise from unwantedneutrophil extravasation and adhesion of the neutrophils to theendothelium. The invention compounds GM 1221, GM 1222, GM 1398 and GM1279 would be used to prevent or treat sepsis.

The effectiveness of these compounds would be shown in a baboon sepsismodel system as described by Taylor et al., J. of Clinical Inv., (1987),79:918, and by Taylor, et al., Circulatory Shock, (1988), 26:227.Briefly, this would consists of determining if the compounds areeffective in treating sepsis by preventing the death, or prolonging thelives of septic animals to either a lethal or sublethal dose of E. coil.A lethal or sublethal dose of E. coil consist of approximately 4×10¹⁰and 0.4×10¹⁰ organisms, respectively. Baboons that receive a lethal doseof E. coil invariably die within 16-32 hours. Taylor, et al., J. ofClinical Inv., (1987), 79:918, and Taylor, et al., Circulatory Shock,(1988), 26:227.

Thus, the procedure would consist of using two administration routinesfor each of the three compounds wherein they are delivered inphysiological saline. In the first, between 1 and 10 mg of compound perkg of body weight is administered in three separate doses at 24, 22, and21 hours before a lethal challenge of bacteria. Alternatively, compoundcan be administered in a single dose simultaneously with the bacterialchallenge. In both instances the compounds would considerably extend thelifetime of the baboons that receive the multiple or single dosetreatment and they would survive well beyond 48 hours.

Example 9 Treatment of Peritonitis

Certain of the invention compounds have been shown to be efficacious intreating peritonitis. The efficaciousness of GM 1221, and GM 1398 wasshown using a murine thioglycollate induced peritonitis model. The assaymaterials and methods are known in the art, or are generally describedby Lewinsohn, D. et al., J. Immun., 138:4313-4321 (1987), or Watson, S.et.al., Nature 349:164-166 (1991).

This assay measures the abili:ty of the compounds to inhibit neutrophilmigation to the peritoneal cavity, the migration being initiated by thepresence of thioglycollate in the peritoneal cavity. Thioglycollate is aknown and effective inflammatory agent that causes neutrophil migrationinto the mouse peritoneum when it is administered intraperitoneally.Lewinsohn, D. et al., J. Immun. (1987) 138:4313-4321.

Briefly, female Swiss Webster mice weighing about 25 grams were injectedin the tail vein with 200 ul of phosphate buffered saline (PBS) with orwithout the appropriate compound. The pH of the solutions was adjustedto neutrality by the addition of either NaOH or HCL and sterilized byfiltration through a 0.2 u filter.

Immediately following injection with the appropriate compound or PBS,the mice were injected intraperitoneally with 1 ml of thioglycollatemedium prepared as described by the manufacturer, BBL. Three hoursfollowing injection of the thioglycollate solution the mice weresacrificed by CO₂ asphyxiation, and the number of cells in theperitoneum removed by lavage with 5 ml of heparinized (5U/ml) 0.9%.sodium chloride solution containing 0.1% bovine serum albumin. Cellnumber was determined using a Coulter Counter. The cells were preparedfor counting by diluting the lavage fluid with 1:50 of a commercialphysiological isotonic solution, Isoton II, and the cells lysed byadding S/P Lysing and Hemoglobin Reagent (1:100 final dilution). Cellnuclei were counted in a sized window with lower and upper limits set at3.9 and 5.7 um, respectively.

FIG. 7 shows the results. It is apparent that both GM 1398 and GM 1221inhibit neutrophil migration into the peritoneum. However, the bettercompound is GM 1398 which inhibited about 30% more neutrophils migrationthan GM 1221.

The instant invention is shown and described herein in what isconsidered to be the most practical and preferred embodiments.Publications listed herein are incorporated herein by reference todisclose specific procedures on how to make, and/or use the invention.Further, it is recognized that departures may be made therefrom whichare within the scope of the invention, and that obvious modificationswill occur to one skilled in the art upon reading this disclosure.

What is claimed:
 1. A method of determining a site of intimation in apatient in need thereof, comprising the steps of:administering to thepatient an effective amount of a compound, wherein the compound has thefollowing structure: ##STR20## wherein m and n are independently aninteger of from 1 to 5, Y and Z are independently a connecting moietyselected from the group consisting of --CH₂ --, --O--, --S--, --NR'--and --NR'R"-- (wherein R' and R" are indepeadently H or an alkylcontaining 1 to 5 carbon atoms); X is a connecting moiety which isselected from the group consisting of --O--, --S--, --NR'--, and acovalent bond; and wherein R'" is selected from the group consistingof 1) hydrogen; 2) an alkyl containing 1 to 4 carbon atoms; ##STR21##wherein R¹ and R² are indepedently an alkyl or an alkenyl group of 1-5or 13-15 carbon atoms; ##STR22## wherein (CH₂)_(n) ' and (CH₂)_(m) ' areindependently a saturated or unsaturated alkyl group of 15 to 24 carbonatoms; ##STR23## wherein R is --CO(CH₂)₁₄ CH₃ ; ##STR24## 9) --OR", SR",--I, --N₃, and NR'R"; wherein A is selected from the group consisting ofα and β forms of sialic acid, Kemp's acid, Quinic acid, Glyceric acid,Lactic acid, acetic acid, --SO₃, --PO₃, and esters thereof; and whereinB is selected from the group consisting of α and β forms of L-fucose,carboxylic acid analogues of fucose, inositol, substituted inositol,benzimidazole, substituted benzimidazole, guanidine, substituted butane,pentaerythritol, and substituted pentaerythritol, wherein thesubstituents are selected from the group consisting of --CH₃, --CH₂ OH,--CH₂ F, and --CH₂ NR³ ₂ wherein each R³ is independently an alkyl of 1to 5 carbon atoms; and ##STR25## wherein Me is a methyl group, R1, R2,and R3 are each independently --OH, --F, and --NR"R" wherein each R" isindependently hydrogen or an alkyl of 1 to 5 carbon atoms; wherein thecompound is attached to a detectable label; allowing the labelledcompound sufficient time to circulate in the patient and attach to aselectin in the patient; and detecting the location of the labelledcompound in the patient, whereby the site of inflammation is determined.2. A method as in claim 1, wherein A is ##STR26## and B is ##STR27## andesters thereof.
 3. A method as in claim 1, wherein A is selected fromthe group consisting of glyceric acid, lactic acid, acetic acid, --SO₃and --PO₃.
 4. A method as in claim 1, wherein the compound is: ##STR28##5. A method as in claim 1, wherein the compound is: ##STR29##
 6. Amethod as in claim 1, wherein the compound is: ##STR30##
 7. A method asin claim 1, wherein the compound is: ##STR31##
 8. A method as in claim1, wherein the selectin receptor is an ELAM-1 receptor.
 9. A method asin claim 1, wherein the detectable label is a radioactive label.
 10. Amethod as in claim 1 wherein:m is 2; n is 2; Y is --O--; Z is --O--; Xis --CH₂ --; R'" is hydrogen; and B is selected from the groupconsisting of α-L-fucose and β-L-fucose.
 11. A method as in claim 10wherein A is N-acetyl neuraminic acid.